Cell culture method and apparatus thereof

ABSTRACT

By rotating a cell-culture container while controlling the temperature and the gaseous atmosphere, cells are cultured under the application of hydrostatic pressure due to centrifugal force, and according to this cell culture, cells are efficiently activated and cultured while inhibiting dedifferentiation.

TECHNICAL FIELD

The invention of the present application relates a method for cellculture and an apparatus thereof. More specifically, it relates to anovel method for cell culture capable of simply and efficientlyobtaining cells specifically useful for regenerative medicine, as wellas an apparatus thereof.

BACKGROUND ART

In cell culture, since during the process to lose the function in a monolayer cell culture growing cells only on the surface layer of a culturemedium cells of a certain type become dedifferentiated, to prevent thisa method of adding growth factors, chemicals, etc. to controldedifferentiation or a method of activating the cell function bymechanical stimulation in conventionally utililzed.

Among the methods of controlling cell dedifferentiation by mechanicalstimulation are a method using hydrostatic pressure (Effects of physicalstimulation on chondrogenesis in vitro, Materials Science andEngineering C6(1998)301-306) and a high density culture techniqueutilizing centrifugal force.

However, existent methods of utilizing hydrostatic pressure have thedrawback that they are not only expensive but also the culture apparatusis large and requires much space. On the other hand, in the existentmethod utilizing the centrifugal force, since the centrifugal force isapplied at a normal temperature after previous culture in an incubator,it involves a problem that it is impossible to have a cultureenvironment continuously applying stimulations for a long time, orperiodically changing the stimulation. In addition, in the existentmethod of utilizing the centrifugal force, control of the temperatureand the surrounding atmosphere was impossible.

In view of the above, it is a goal of the invention of the presentapplication to solve the problems described above and to provide a newmethod capable of simple and efficient cell culture while suppressingdedifferentiation mechanically by stimulations, as well as an apparatusthereof.

DISCLOSURE OF THE INVENTION

The invention of the application, in order to solve the foregoingproblems, provides firstly a method for cell culture which continuouslycontrols dynamic conditions by application of hydrostatic pressure on aculture liquid in a condition for culturing cells by centrifugal force,thereby giving stimulation to the cells. Secondarily, it provides amethod for cell culture wherein for the controlling of dynamic culturecondition by application of hydrostatic pressure, application of thehydrostatic pressure to the cells is periodically changed or maintainedfor a certain period of time by application of centrifugal force.Thirdly, it provides a method for cell culture as described abovewherein the hydrostatic pressure is applied in a range of 60 MPa orless; fourthly, it provides a method for cell culture described abovewherein the hydrostatic pressure is applied within a range from 0.5 secto 6 weeks, fifthly, it provides a method for cell culture describedabove wherein application of the hydrostatic pressure is conductedthrough control of the number of rotation of a centrifugator and,sixthly, it provides a method for cell culture described above whereinthe temperature and the atmosphere are controlled. Then, seventhly, theinvention of the present application provides a method for cell cultureas described above wherein the cells are cultured together with variouskinds of biomaterials.

Further, eighthly, the invention of the present application provides anapparatus for the cell culture having a cell-culturing device supportedby a rotational shaft in a sealed container for providing cells with ahydrostatic pressure by centrifugal rotation, ninthly, it provides anapparatus for the cell culture as described above, wherein comprising acontrol mechanism for controlling the rotation time and the rotationspeed of the cell culturing device, tenthly, it provides an apparatusfor the cell culture as described above wherein the number of rotationsis controllable within a range from 10 to 25000 rpm for providing ahydrostatic pressure at 60 MPa or less and, eleventhly, it provides anapparatus for the cell culture as described above wherein the inside ofthe cell culturing device is divided such that a plurality types of cellcultures can be conducted simultaneously.

The invention of the application provides, twelfthly, an apparatus forthe cell culture as described above wherein an injection port and anexhaust port for an atmospheric gas into and out of the sealedcontainer, and a control mechanism for injecting and exhausting theatmospheric gas are provided, and, thirteenthly, it provides anapparatus for the cell culture as described above wherein a controlmechanism for the temperature in the sealed container is provided.

In the growth of organisms, stem cells, which are undifferentiated andnot specified as to function, are successively specialized with regardto function while repeating differentiation into various organs ortissues. However, the cell differentiation in the organism is not alwaysconducted under identical conditions, circumstances such as the pressureor the temperature in the organism undergoing change.

The invention of the application takes this point into account and isintended to culture cells under conditions approximating actual in-vivoconditions. The gist of the invention of the application intending tocreate an environment similar to the organism has a feature of culturingthe cells while controlling culture environment by the centrifugalapplication of a hydrostatic pressure as described above.

Such a feature is based on the knowledge that the liquids inside aspecimen tube rotationally driven by a centrifugal force always receivea centrifugal force and a hydrostatic pressure, the level of thecentrifugal force and the hydrostatic pressure can be controlled simplyby controlling the number of rotations of a centrifugator, and that itis possible to give stimulations to the cells and suppress celldedifferentiation by executing this control during culture of the cells.

Further, the invention of the application also achieves control of thetemperature and the atmosphere in the culture. The invention accordingto the application, enables growth of cells while controlling activationand dedifferentiation as described above and makes it possible to obtainin a short period of time various kinds of organism tissues includingthe extra-cellular matrix extending between cells, which maintainshealth of cells and in which is written information to suppress growthand dedifferentiation. This is promising for application in regenerativemedicine.

For example, by periodically applying hydrostatic pressure of from 0.1MPa to 30 MPa to the cells by rotating the cell culture apparatus of theinvention, not only the in-vivo circumstance equal to the rhythm ofhuman walking but also can control high gravitational force (forexample, as in the environment in the deep sea). Then, by periodicallychanging hydrostatic pressure and the centrifugal force on the cells,thereby enhancing the cell activity, cell agglomerates are formed, andthe hydrostatic pressure controls the cell metabolism to control thededifferentiation.

The features as described above cannot be attained by or even inducedfrom the existent methods of utilizing the hydrostatic pressure or theexistent methods of exerting the centrifugal force after previouslyconducting the culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross sectional constitutional view exemplifying theoutline of an apparatus for the cell culture according to the inventionof the present application.

FIG. 2 shows a schematic view exemplifying a test container in a cellculturing device and an exerted force.

FIG. 3 shows a correlation graph between hydrostatic pressure and timeexemplifying the state of keeping an identical hydrostatic pressure.

FIG. 4 shows a correlation graph between hydrostatic pressure and timeexemplifying the state of periodically changing the hydrostaticpressure.

FIG. 5 shows a graph showing the increase for the number of cells in theinvention utilizing centrifugal force of the present application and ina conventional high density culturing method and mono-layer culturingmethod.

FIG. 6 shows a photograph by a phase-contrast microscope of cellscultured for one week by a method of applying a hydrostatic pressure ofthe invention according to the present application.

FIG. 7 shows a photograph by a phase contrast microscope for cellscultured for 1 week by a conventional mono-layer culturing method.

References in the drawings shown the followings.

1 . . . cell-culturing device

2 . . . lid for the cell-culturing device

3 . . . rotational shaft

4 . . . motor

5 . . . heater

6 . . . sealed container

7 . . . culturing tube

8 . . . distance between a rotary shaft and a culture solution surface

9 . . . centrifugal force

C . . . control mechanism

G . . . carbon dioxide-incorporated steam

BEST MODE FOR CARRYING OUT THE INVENTION

The invention of the present application has features as described aboveand embodiments thereof are to be described below.

At first, referring to a method for cell culture and the outline of anapparatus according to the invent ion of the present application withreference to the drawings, FIG. 1 shows an overall view of a cellculturing apparatus in which cells and a culturing liquid can be freelyfilled and taken out by opening and closure of a lid (2). Thecell-culturing device (1) is supported in a sealed container (6) havinga heating mechanism (5), by a rotational shaft (4) connected with amotor (3). Further, steam (G) incorporated with gaseous carbon dioxideis circulated to the sealed container (6). The atmospheric gas isinjected by way of a channel formed in the lid (2) into thecell-culturing device (1) and exhausted therefrom by the opening andclosure of the lid (2). Then, the rotational speed, the rotational time,and the temperature and the atmospheric gas for the cell-culturingdevice (1) are controlled by a previously set control mechanism (C).

FIG. 2 is a schematic view partially exemplifying the inside of thecell-culturing device (1) of FIG. 1, in which a hydrostatic pressure canbe applied by centrifugal force (9) to the cells in the container (7) ofa test tube-like configuration connected with the rotational shaft (3),created by rotating the same after charging cells and culturing liquid.

The rotational speed and the rotational time in this case are controlledby the control mechanism (C) in FIG. 1.

Thus, the level of the hydrostatic pressure, the centrifugal forceexerted on the cells, and the time thereof can be freely controlled.

The level of the hydrostatic pressure and the centrifugal force arerepresented, for example, as shown below. TABLE 1 Hydrostatic pressureCentrifugal Force depends on the height of solution (exerted on cell ·material) HP (atm cm²/Kg) = 5.48 × RCF(G) = 11.18 × r(cm) × 10⁻⁹ × D ×Q² (r² − r _(men) ²) (Q/1000)² D: Average density of r: distance betweengranules solution (g ml⁻¹ and center r: distance from the Q: rotationalspeed (rpm) center (cm) rmem: distance from the liquid surface to thecenter of rotation (cm)

The relation between the number of rotations and the hydrostaticpressure is exemplified as below. TABLE 2 Cell culture liquid 1.14g/cm⁻¹ (MEM) Height of solution 2 cm Number of rotation 3000 rpm 5000rpm 10,000 rpm Hydrostatic pressure 2 Mpa 5.6 Mpa 22.5 Mpa

FIG. 3 shows the hydrostatic pressure in the container (7) when therotational speed is maintained for a certain period and FIG. 4 shows thechange of the hydrostatic pressure in the container (7) when therotational speed is changed intermittently. The invention of the presentapplication is conducted due to application patterns of the hydrostaticpressure in the container which are shown in a simplified manner in FIG.3 and FIG. 4 or combination of such application patterns, and thesetting of the conditions for this combination is based on the followingfacts.

The specific number of rotations for forming the hydrostatic pressure inthe case of culturing similar to in-vivo conditions is, preferably, from10 to 25,000 rpm and particular, preferably from 10 to 23,000 rpmbecause when the number of rotation exceeds 25,000 rpm, the hydrostaticpressure exceeds 60 Mpa and possibly destroys the cells.

Further, the application time of the hydrostatic pressure is limited toa range of from 0.5 sec to 6 weeks. This is because the cycle ofpressure exerted on a human body during vigorous motion is considered tobe 0.5 sec and on the other hand, the culturing period of cells usedupon transplantation to a human body is about within four weeks.

Moreover, the temperature is preferably from about 0° C. to 50° C. whichis within the range of living organisms, and a temperature about from25° C. to 40° C. which is the usual body temperature of organisms isparticularly preferred.

FIG. 5 shows comparison between the cell culture under the conditionsdescribed above and cell culture according to a conventional method.FIG. 5 shows those cultured according to the invention of the presentapplication (shown by solid circles), those grown by conventional highdensity culture (indicated by blank squares), and those of mono-layerculture (indicated by blank circles), cultured for 1 week to 3 weeks,the number of cells being plotted. As is apparent from FIG. 5, the cellgrowth ratio in the invention of the present application is 15% greaterthan the existent high density culture and 30% greater than the existentmono layer culture after three weeks.

Examples of the invention according to the application and the monolayerculture are shown below and described more specifically.

Off course, the invention is not restricted by the following examples.

EXAMPLE

Cartilage cells (5×10⁴ cells/ml) were grown by centrifugal culture in aculture solution of MEM (Minimum Essential Medium) at a temperature of25° C. and in atmospheric air for 1 week. The centrifugal conditionswere: rotating for 30 min twice a day at a 1000 rpm, conducted threetimes per week. On the other hand, a monolayer culture was conducted asa control.

As a result of centrifugal culture, the final number of cells was10.2×10⁴ cells/ml, whereas it was 8.4×10⁴ cells/ml in the monolayerculture; thus the centrifugal culture improves the growth ratio by atleast 20%.

Further, FIG. 6 and FIG. 7 show the states in comparison by phasecontrast microscopic photograph.

It is clearly shown that the number of cells grown by the method forcentrifugal culture according to the invention of the presentapplication (FIG. 6) is greater than the number of cells grown by themono layer culture method (FIG. 7). Further, spherical shapes inherentto the cartilage cells remain in the centrifugal culture, whereas theywere dedifferentiated into cells of advanced fibrillation in themonolayer culture.

From the foregoing result, it was confirmed that the centrifugal culturecan increase the growth ratio of cells and also suppressdedifferentiation of cells.

INDUSTRIAL APPLICABILITY

As has been described above in detail, the invention of the presentapplication not only can efficiently manufacture substrate materials forthe cell growth in regenerative medicine by activating the cells andsuppressing dedifferentiation but also can grow, conveniently andefficiently, specific cells for the individual patients in so-calledtailor-made treatment.

1-13. (canceled)
 14. A method for cell culture which continuouslycontrols dynamic conditions by application of hydrostatic pressure on aculture liquid in an atmospheric air condition or a gaseous atmospherecondition for culturing cells by centrifugal force, thereby givingstimulation to the cells.
 15. A method for cell culture according toclaim 14, wherein for the controlling of dynamic culture conditions byapplication of hydrostatic pressure, application of the hydrostaticpressure to the cells is periodically changed or maintained for acertain period of time by application of centrifugal force.
 16. A methodfor cell culture according to claim 14, wherein the hydrostatic pressureis applied in a range of 60 MPa or less.
 17. A method for cell cultureaccording to claim 14, wherein the hydrostatic pressure is appliedwithin a range from 0.5 sec to 6 weeks.
 18. A method for cell cultureaccording to claim 14, wherein the application of the hydrostaticpressure is conducted by controlling the number of rotations of acentrifugator.
 19. A method for cell culture according to claim 14,wherein the temperature and the atmosphere are controlled.
 20. A methodfor cell culture according to claim 14, wherein cells are culturedtogether with various kinds of biomaterials.
 21. An apparatus for cellculture having a cell-culturing device supported by a rotational shaftin a sealed container for providing cells with hydrostatic pressure bycentrifugal rotation.
 22. An apparatus for cell culture according toclaim 21, wherein comprising a control mechanism for controlling therotation time and the rotation speed of the cell-culturing device. 23.An apparatus for cell culture according to claim 21, wherein the numberof rotations is controllable within a range from 10 to 25,000 rpm forproviding the hydrostatic pressure of 60 MPa or less.
 24. An apparatusfor cell culture according to claim 21, wherein the inside of the cellculturing device is divided so that a plurality of types of cells can becultured simultaneously.
 25. An apparatus for cell culture according toclaim 21, wherein an injection port and an exhaust port for the gaseousatmosphere into and out of the sealed container, and a control mechanismfor injecting and exhausting the atmospheric gas are provided.
 26. Anapparatus for cell culture according to claim 21, wherein a controlmechanism for the temperature in the sealed container is provided.
 27. Amethod for cell culture according to claim 15, wherein the hydrostaticpressure is applied in a range of 60 MPa or less.
 28. A method for cellculture according to claim 15, wherein the hydrostatic pressure isapplied within a range from 0.5 sec to 6 weeks.
 29. A method for cellculture according to claim 16, wherein the hydrostatic pressure isapplied within a range from 0.5 sec to 6 weeks.
 30. A method for cellculture according to claim 27, wherein the hydrostatic pressure isapplied within a range from 0.5 sec to 6 weeks.
 31. A method for cellculture according to claim 15, wherein the application of thehydrostatic pressure is conducted by controlling the number of rotationsof a centrifugator.
 32. A method for cell culture according to claim 16,wherein the application of the hydrostatic pressure is conducted bycontrolling the number of rotations of a centrifugator.
 33. A method forcell culture according to claim 27, wherein the application of thehydrostatic pressure is conducted by controlling the number of rotationsof a centrifugator.